Milk fat (MF) triacylglycerols (TAG) determine the physical and functional properties of butter and products rich in MF. To predict these properties, it is necessary to understand the variability of fatty acids, TAG, their associations, and their effect on milk productive traits, days in milk (DIM), and genes related to fat synthesis. Therefore, the aim of this research was to study the variability of TAG using MF from individual cows and to investigate the effect of fatty acid composition, DGAT1 K232A polymorphism, DIM, and milk production traits (fat content and morning milk yield) on MF TAG profile in the Dutch Holstein-Friesian dairy cattle population. Large differences in MF TAG profiles were seen among cows. We showed that the variability of TAG is highest for low and high molecular weight TAG [TAG with carbon numbers (Cn) 26-30 and Cn52-54, respectively] and lowest for TAG with Cn38, which was the most abundant TAG. Saturation index (saturated fatty acid/unsaturated fatty acid) and the ratio C16:0/C18:1 cis-9 showed significant effects on TAG Cn34, Cn36, Cn52, and Cn54: TAG Cn34 and Cn36 increased as the saturation index and ratio C16:0/C18:1 cis-9 increased, whereas the opposite was seen for TAG Cn52 and Cn54. Moreover, the DGAT1 K232A polymorphism significantly affected TAG Cn38. We showed that the relative concentration of TAG with Cn38 was higher in cows with DGAT1 KK genotype. Production traits (fat content and morning milk yield) and DIM had no significant effect on TAG profile. This is a relevant observation because considerable increases of milk yield and fat content have been seen in the Netherlands over the last 60 yr. The large differences shown between individual cows in MF TAG profile imply differences in physical properties of MF.